Infectious Bronchitis Virus (IBV)
Avian infectious bronchitis virus (IBV) is a highly infectious and contagious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct. IBV is a member of the Coronaviridae and genetically very similar coronaviruses cause disease in turkeys and pheasants.
Clinical signs of IB include sneezing, tracheal rales, nasal discharge and wheezing. Meat-type birds have reduced weight gain, whilst egg-laying birds lay fewer eggs. The respiratory infection predisposes chickens to secondary bacterial infections which can be fatal in chicks. The virus can also cause permanent damage to the oviduct, especially in chicks, leading to reduced egg production and quality; and kidney, sometimes leading to kidney disease which can be fatal.
Both live and attenuated vaccines are currently used in IB vaccination. To date, the most efficacious vaccines are live attenuated viruses empirically produced following blind repeated passages through embryonated eggs.
A problem with this approach is that, upon serial passaging, the immunogenicity of the virus decreases. It is necessary to achieve a balance between an acceptable degree of attenuation to make the virus safe, and an acceptable loss of immunogenicity such that the virus vaccine is still efficacious. This “balancing” of attenuation is a trial and error approach, rendering the outcome of the attenuation process uncertain.
Since attenuation by serial passage is effectively a random event, the resultant vaccine is ill-defined genetically as the molecular basis of the attenuation is unknown. Each batch of attenuated virus will be different, making it difficult to achieve consistency of the resulting vaccine and reproducibility of the protective/therapeutic effect in vivo.
A further disadvantage is that embryonated eggs are expensive and cannot be used as a prolonged source of virus.
Growth of virus on embryonated eggs is a cumbersome process as each egg must be sterilized, candled, inoculated with virus and incubated before harvesting small volumes of allantoic fluid from each egg and pooling before purification. The lack of reliable supplies of high quality eggs results in limitations in the amount of vaccine which may be produced, particularly in an emergency situation.
In addition to these logistic and supply problems, embryonated eggs have other limitations as a host system for vaccine production. For example, there are increasing concerns about the presence of adventitious viruses, particularly retroviruses in eggs, which would compromise the production of live, attenuated viral vaccines.
There is therefore a need for alternative IBV vaccines and methods for their production which do not suffer from the above mentioned drawbacks.
Coronaviruses
Coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, single-stranded, positive sense RNA genome of 27 to 32 kb.
All coronavirus lipid envelopes contain at least three membrane proteins: the spike glycoprotein (S), integral membrane protein (M), and small membrane protein (E). The coronavirus S protein is a type I glycoprotein which oligomerizes in the endoplasmic reticulum and is assembled into virion membranes through non-covalent interactions with the membrane protein. Following incorporation into coronavirus particles, the S protein is responsible for binding to the target cell receptor and fusion of the viral and cellular membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; the ectodomain, which is present on the outside of the virion particle; the transmembrane region responsible for anchoring the S protein into the lipid bilayer of the virion particle; and the cytoplasmic tail.
The IBV S protein (1,162 amino acids) is cleaved into two subunits, S1 (535 amino acids; 90 kDa) comprising the N-terminal half of the S-protein, and S2 (627 amino acids; 84 kDa) comprising the C-terminal half of the S protein.
The S2 protein subunit associates non-covalently with the S1 subunit and contains the transmembrane and C-terminal cytoplasmic tail domains.
The S1 subunit has been widely reported to comprise the receptor-binding activity of the S protein.
For example, it has been shown for the serogroup I coronavirus, human coronavirus HCoV-229E, that of three variants having truncation in the N-terminal domain of S1, two were unable to bind the receptor, implicating the region between amino acids 417 and 547 as important for receptor binding (Bonavia et al (2003) J. Virol 77. 2530-2538).
The first 330 amino acids of the 769-residue S1 subunit of the mouse hepatitis virus (MHV) S protein are sufficient to bind the MHV receptor (Kubo et al (1994) J. Virol. 68:5403-5410). Similarly an 193-amino acid fragment of the SARS S protein (residues 318-510) binds to the receptor and blocks S-protein mediated infection (Wong et al (2004) J. Biol. Chem. 279:3197-3201).
It is also reported that amino acids 1-510 of the SARS-CoV S glycoprotein represent a domain containing the receptor binding site (amino acids 270-510) analogous to the S1 subunit of other coronavirus S glycoproteins (Babcock et al (2004) J. Virol. 4552-4560).
The S protein is a determinant of the cell tropism of the virus (Casias et al (2003) J. Virol. 77:9084-9089). It has been shown that amino acid substitutions in the N-terminal region of the S1 protein are associated with the extended host range of a virus variant of murine hepatitis virus (MHV) (Thackray and Holmes (2004) Virology 324:510-524). Moreover, it is generally thought that species specificity of infection is due to the specificity of the virus-receptor interaction (Compton et al (1992) J. Virol. 7420-7428; Gagneten et al (1995) J. Virol. 69:889-895).
It has, to date, thus been widely assumed that cell tropism is a property of the S1 domain of the S protein of coronaviruses.